The invention relates A novel marine microorganism (Pseudomonas CH07) isolated from the Indian coastal zone near Goa which is capable of biodegradation of PCBs including sterically hindered di and tri-ortho chlorinated biphenyls and coplanar congeners present in a technical grade Clophen A-50 (Bayer, Lot no. 16572) by the novel strain of marine microorganism, Pseudomonas CH07.
Classification of PCBs
PCBs are non-volatile organic compounds (depending on the degree of chlorination they may belong to the class of semi volatile organic compounds (SVOC). PCBs are divided into two groups of isomers based on the difference in structure:
1. Coplanar PCBsxe2x80x94those compounds have chlorinated substituents in both para positions, and any/all meta positions. Meta or para chlorine substituents have, by their structure, low steric hindrance with neighboring H, which allows free rotation about the phenyl-phenyl bond. There are 20 coplanar PCBs, out of which three (77, 126, 105) are very toxic. Most importantly, they are non-ortho chlorinated.
2. Mono-ortho chlorinated PCBs-all molecules have one-chloro substitutions in the other positions only. Ortho substituents tend to create rigid bonds due to the large steric interference between C1 and H atoms.
3. Di-ortho a represents the chlorine atoms at the ortho-positions.
4. Tri-ortho represents the chlorine atom at the ortho-positions.
Depending on the number of C1 atoms on the biphenyl rings PCBs are divided into mono, di, tri, tetra, penta, hexa, hepta, octa, nona and deca-chlorobiphenyls and there are 209 possible PCBs theoretically but many do not occur because of steric hindrance.
Most abundant PCBs in commercial mixtures are ortho-substituted congeners, which are readily degradable. However, smaller amounts of the so-called xe2x80x98di-oxinxe2x80x99 like PCBs namely the coplanar (=non-ortho substituted) and mono-ortho substituted congeners, are present in the commercial mixture as well. Broad classification:
a) Non-ortho or mono-ortho chlorinated Biphenyls (coplanar).
b) Ortho-substituted chlorinated biphenyls.
c) Sterically hindered chlorinated Biphenyls.
Polychlorinated biphenyls (PCBs) represent a class of toxic xenobiotics that are distributed throughout the biosphere. Over the past several years, PCBs have received increasing attention due to accumulation of their residues in tissues of living organisms and biomagnified through the food chain leading to health hazards. PCBs are produced by direct chlorination of biphenyl. Due to the large number of hydrogen atoms present on the biphenyl nucleus, many different chlorinated compounds (termed xe2x80x9ccongenersxe2x80x9d isomers of different homologous series) are possible. As many as 209 congeners of the PCBs could be theoretically produced [Furukawa, Biodegradtion and Detoxification of Environmental Pollutants, p. 34-57. CRC press. (1982)]; however, due to steric restrictions, only about half of this number are actually found in the environment. Therefore, PCBs are mixtures of a variety of chlorine-substituted biphenyl molecules. Clophen A-50 is a technical grade chemical compound containing about 40 different congeners of PCBs. Clophen is well known in commercial circles and to peoples skilled in the art. In fact, Clophen A-50 is equivalent to Aroclor 1256 i.e. it has about 56% chlorines w/w [Yadav; Jagjit S.; Reddy; C. A.; Quensen; John F.; Tiedje; James M. Degradation of polychlorinated biphenyl mixtures in soil using Phanerochaete chrysosporium in nutrient rich, non-ligninolytic conditions. U.S. Pat. No. 6,107,079. (Aug. 22, 2000)].
Due to their lipophilic and hydrophobic characteristics, the PCBs get accumulated in tissues of various species of organisms and are magnified through the food chain [Furukawa supra; Jacobson et al. Develop. Psychol. 20: p.523-532. (1984); Sarkar, A. and Everaarts, J. M. (1998) Riverine input of chlorinated hydrocarbons in the coastal Pollution. In: Ecology of Wetlands and Associated Systems. Ed. S. K. Majumdar, E. W. Miller and Fred J. Brenner. Chapter 27, Pub: Pennsylvania Academy of Science. pp, 400-423; Sarkar, A. (1994) Occurrence and distribution of persistent chlorinated hydrocarbons in the seas around India. In: The Oceans: Physico-chemical Dynamics and Resources (ed) S. K. Majumdar, E. W. Miller, G. S. Forbes, R. F. Schmalz and Assad, A. Panah. The Pennsylvania Academy of Science. Chapter-28,pp, 445-459.]. The physical effects of PCBs vary from mammals, to birds, to humans. Natural microbial populations do not easily remove PCBs. Some PCB congeners are found to be transformed by both anaerobic and aerobic bacteria [Abramowicz, D. A., Crit. Rev. Biotechnol. 10: 241-251. (1990)]. The aerobic degradation of PCBs is generally limited to lessxe2x80x94chlorinated congeners (five or fewer chlorines per biphenyl molecule) by an enzymatic mechanism involving deoxygenation of the aromatic ring [Bedard, D. L., et al., Appl. Environ. Microbiol. 53: 1094-1102. (1987); Bradley; Clifford A.; Kearns; Robert D.; Wood; Pauline P.; Black; William E. Degradation of polyhalogenated biphenyl compounds with white-rot fungus grown on sugar beet pulp. U.S. Pat. No. 5,583,041 (Dec. 10, 1996); Sarkar, A. (1994) Comments on xe2x80x9cDegradation of polychlorinated dibenzo-p-dioxin and dibenzo-furan contaminants in 2,4,5-T by photoassisted iron-catalyzed hydrogen peroxidexe2x80x9d by J. J. Pignatello and L. Q. Huang. Wat. Res. Vol. 27: 1731-1736. Water Research. Vol: 28 No. 12, pp, 2589-2594. Sarkar, A. (1994) Comments on: xe2x80x9cEvaluation of dechlorination mechanisms during anaeorobic fermentation of bleached kraft mill effluentxe2x80x9d, by W. J. Parker, E. R. Hall and G. J. Farquhar, Wat. Res. 27, 1269-1273 (1973). Water Research Vol. 28: No. 9, pp, 2043-2044, 1994.] The more chlorinated congeners are generally recalcitrant to aerobic degradation [Kimbara; Kazuhide; Shimura; Minoru; Hatta; Takahasi; Kiyohara; Hohzoh. Method for degrading polychlorinated biphenyls and novel microorganism. U.S. Pat. No. 5,897,996 (Apr. 27, 1999)]. In 1978, Furukawa and associates [Furukawa et al., Appl.Environ. Microbiol. 35:223-227. (1978)] studied the biodegradability of several isomers of PCBs. They found that as chlorine substitution increased, degradability decreased. An isomer with four Cl was not easily degraded. The position of the chlorine is also important. Ortho positioning of two chlorines on a single ring greatly inhibited degradation [MacFaddin, F. J. 1980. Biochemical tests for identification of medical bacteria. Second edition. p. 527. Williams and Wilkins, Baltimore].
Considering the environmental importance of PCBs and the hazards posed by them, numerous investigators have been examining biological detoxification systems to deal with PCBs. One way to decipher the complexities of highly chlorinated isomers is to look into the problem, according to the order of the extent of chlorine substitution in the biphenyl ring. Of the 209 theoretically possible isomers and congeners of PCB, 20 members attain coplanarity due to non-ortho chlorine substitution in the biphenyl rings. In this group, three coplanar congeners such as 3,3,4,4xe2x80x2-tetrachlorobiphenyl, 3,3xe2x80x2,4,4xe2x80x2,5-pentachlorobiphenyl and 3,3xe2x80x2,4,4xe2x80x2-pentachlorobiphenyls and 3,3xe2x80x2,4,4xe2x80x2,5,5xe2x80x2-hexachlorobiphenyls are approximate isostereomers of highly toxic 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,4,7,8-pentachlorodibenzofuran and hence elicit similar toxic and biologic responses typical of dioxins and furans [Safe, S.(1984) Polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs): biochemistry, Toxicology and mechanism of action. CRC Crit. Rev. Toxicol., 13, 319-93]. 
Because of the coplanarity, these three congeners of PCB were found to be most toxic chlorinated aromatic compounds in comparison with the rest of the congeners of PCBs [Yoshihara, S.; Nagata, K.; Yoshimura, H.; Kuroki, H. and Masuda, Y. (1981). Inductive effect on hepatic enzymes and acute toxicity of individual polychlorinated dibenzofuran congeners in rats. Toxicol. Appl. Pharmacol., 59, 580-588]. Several earlier studies indicate that toxic nature of technical PCB mixtures may be associated with the presence of trace levels of particular toxic PCB congeners having four or more chlorine atoms at both para and meta positions in the biphenyl rings but no chlorine atoms in ortho positions. [Yoshilhara, H., Ozawa, N. and Saeki, S. (1978). Inductive effect of polychlorinated biphenyl mixture and individual isomers on the hepatic microsomal enzymes. Chem. Pharmacol. Bull. (Tokyo), 26, 1215-21]
From structural point of view the physico-chemical properties of different conngeners of PCBs vary according to the positions of chlorine atoms in the biphenyl ring. 
The substitution of chlorine atoms at the ortho positions (2,2xe2x80x2 and 6,6xe2x80x2) of the biphenyl rings clearly indicate the steric hindrance between them due to which the two aromatic rings of the biphenyl get distorted into non-planar configuration as shown below. 
PCBs have widely been used industrially largely because of their versatile characteristics such as non-inflammability, hydrophobicity, thermal stability etc. Such physico-chemical properties of PCBs have made it highly useful as heat resistant compounds in the transformer and in other high-temperature applications. PCBs have also been used in plasticizers, heat transfer and capacitor systems, surface coatings, printing inks, carbonless duplication paper, and waxes [Barton and Marlene, R. Bacterial degradation of 4-chlorobiphenyl. U.S. Pat. No. 4,999,300. (Mar. 12, 1991)]. Clophen A-50 is a technical grade chemical compounds containing different congeners of PCBs. Clophen A-50 is almost equivalent to Aroclor 1256 (i.e. about 56% chlorines w/w). While industrial use of PCBs has been sharply restricted, significant quantities of PCBs are still being released into the environment from waste dumps [25. Sarkar, A. Shailaja, M. S. and Desa, E. (2000) Analysis of PCBs in waste oil samples. Sponsored (Central Pollution Control Board, New Delhi) Project Report no. NIO/SP-23/2000; Sarkar, A. Shailaja, M. S. and Desa, E. (1999) Analysis of PCBs in Environmental samples. Sponsored (Central Pollution Control Board, New Delhi) Project Report no. NIO/SP-21/99] and failure of old electrical equipment. PCB contamination has been observed in drinking water sediments [Boon, J. P., Everaarts, J. M., Kastoro, W. W., Razak, H., Sumanta, I., Sumarno, Nelissen, P. H., Stefels, J. and Hillebrand, M. Th. J. (1989). Cyclic organochlorines in epibenthic organisms from coastal water around East Java, Neth. J. of Sea Res. 23: 4, 427439. Boons, J. P., Van Zantvroot, M. B., Govaert, M. J. M. A. (1985). Organochlorines in benthic polychaetes (Nephtys spp.) and sediments from the southern North Sea. Identification of individual PCB components, Neth. J. of Sea Res. 19:2, 93-109], wastewater, foods and aquatic organisms and especially in fish.
The main object of the invention is to provide a novel marine bacterium Psedumonas CH07 which was deposited on Jul. 9, 2002 in the ARS Patent Culture Collection, Microbial Genomics and Bioprocessing Research Unit, National Center for Agricultural Utilization Research, 1825 N. University Street, Peoria, Ill., 61604, as NRRL B-30604, which strain having degrading properties of congeners and has been isolated from coastal zone of Arabian Sea.
Another object of the invention is to provide the stain capable of sustaining growth in medium containing 100-ppm final concentration of Clophen A-50. Still another object of the invention is to provide a microbial process for the degradation of PCBs present in Clophen A-50 using the marine bacterium. Yet, Another object of the invention is the degradation of congeners present in the Clophen A-50 using the marine bacterium Psedumonas CH07. Yet, another object of the invention is to provide a process for degradation of non-ortho or mono-ortho chlorinated biphenyls (coplanar), ortho-substituted chlorinated biphenyls and sterically hindered chlorinated biphenyls PCBs present in Clophen A-50 using the novel marine bacterium Psedumonas CH07.
The applicants have isolated a marine bacterium of the genus Pseudomonas that is cable of degrading several congeners of chlorine content (4-7) to lesser chlorine containing congeners from a technical grade PCBs (Clophen A-50). The present invention comprises a biologically pure strain of the genus Pseudomonas having the characteristics stated in the table1. The present invention is used to degrade the technical grade PCBs (Clophen A-50) and similar kind of chemicals for biodegradation of deadly PCBs. This strain is capable of sustaining growth in medium containing 100-ppm final concentration of Clophen A-50.
Accordingly, the present invention provides a novel marine bacterium, Pseudomonas CH07, which was deposited in the depository at National Institute of Oceanography, Dona Paula, Goa 403004, India, and available to the public since Apr. 2, 2000, which strain is used for degradation of several/different congeners of PCBs contained in Clophen A-50 based on its unique properties.
The novel strain of marine microorganism designated as Pseudomonas CH07 is capable of degrading several congeners of PCB namely tetra-chlorobiphenyls, pentachlorobiphenyls, hepta-chlorobiphenyls containing 4-7 chlorine atoms on the biphenyl ring, sterically hindered di, tri-ortho chlorinated biphnyls and coplanar congeners of PCB.
In an embodiment of the present invention, the bacterium degrades non-ortho or mono-ortho chlorinated biphenyls (coplanar).
In another embodiment of the present invention, the bacterium degrades ortho-substituted chlorinated biphenyls.
In another embodiment of the present invention, the bacterium degrades sterically hindered chlorinated biphenyls.
In still another embodiment of the present invention, the bacterium degrades most of the congeners present in Clophen A-50 either its single or multiple congeners.
In an embodiment of the present invention, the bacterium degrades the congeners of PCB are selected from tetra-chlorobiphenyls, pentachlorobiphenyls, hexachloro biphenyls and hepta-chlorobiphenyls containing 4-7 chlorine atoms on the biphenyl ring, sterically hindered di, tri-ortho chlorinated biphenyls and coplanar congeners of PCB.
Yet another embodiment of the invention, the bacterium is used to detoxify Clophen A-50 and its constituent congeners of PCB when present at or equal to 100 ppm level.
Yet another embodiment of the invention, the bacterium is capable of degrading six sterically hindered di-ortho chlorinated biphenyls (cb-101, cb-97, cb-141, cb-138, cb-128 and cb-180).
Yet, another embodiment of the invention, the bacterium is capable of degrading two of the three most toxic coplanar PCBs in Clophen A-50.
Yet another embodiment of the invention, the bacterium degrades the PCBs present in Clophen A-50 within a short period of 40 hours.
Yet another embodiment, the bacterium culture used to degrade toxic PCBs present in Clophen A-50 is in the range between10 xcexcl to 50 xcexcl.
Yet another embodiment, the bacterium degrades 20 to 100% of PCBs present in Clophen A-50.
In one more embodiment of the present invention, the microbial process for the degradation of PCBs present in Clophen A-50 comprising culturing the marine bacterium Pseudomonas CH07 and treating the PCBs with the bacterium for degrading congeners present in Clophen A-50.
In yet another embodiment, the bacterium Pseudomonas CH07 degrades non-ortho or mono-ortho chlorinated biphenyls (coplanar), ortho-substituted chlorinated biphenyls and sterically hindered chlorinated biphenyls PCBs present in Clophen A-50.
In yet another embodiment, PCBs are degraded substantially within a short period of 40 hours.
In yet another embodiment, the congeners of PCB degraded are selected from tetra-chlorobiphenyls, pentachlorobiphenyls, hexachlorobiphenyls and hepta-chlorobiphenyls containing 4-7 chlorine atoms on the biphenyl ring, sterically hindered di, tri-ortho chlorinated biphenyls and coplanar congeners of PCB.
In yet another embodiment, tetra chloro biphenyls present in Clophen A-50 is degraded by this bacterium.
In yet another embodiment, seven pentachloro biphenyls present in Clophen A-50 are degraded.
In yet another embodiment, four hexachlorobiphenyls present in Clophen A-50 are degraded.
In yet another embodiment, two heptachlorobiphenyls present in Clophen A-50 are degraded.
In yet another embodiment, the bacterium degrades the PCBs at a concentration ranging between 10 xcexcl to 50 xcexcl.
In yet another embodiment, the bacterium degrades 20 to 100% of PCBs present in Clophen A-50.
In yet another embodiment, the bacterium is used to detoxify Clophen A-50 and its constituent congeners of PCB when present at or equal to 100-ppm level.
In yet another embodiment, the bacterium is isolated from marine environment and not altered genetically, and can be employed in situations at experimental pilot and at commercial scales, wherever there are marine conditions.
In yet another embodiment, the bacterial strain is capable of degrading six sterically hindered di-ortho chlorinated biphenyls (cb-101, cb-97, cb-141, cb-138, cb-128 and cb-180).
In yet another embodiment, two sterically hindered tri-ortho chlorinated biphenyls (cb-151 and cb-181) are degraded.
In yet another embodiment, the sterically hindered congeners are degraded by this organism under marine condition.
In yet another embodiment, different congeners of PCBs present in Clophen A-50, two congeners (CB-126, CB-181) get completely degraded.
In yet another embodiment, the congener cb-126 is completely degraded in about 40 hours.
In yet another embodiment, the marine bacterium is the most potent for degradation of two of the three most toxic coplanar PCBs in Clophen A-50.
In yet another embodiment, the organism is capable of degrading PCB congeners, cb-126 and cb-77 under marine condition.
In yet another embodiment, the coplanar PCB (3,3xe2x80x2,4,4xe2x80x2-tetrachlorobiphenyl) is degraded very substantially within a short period of 40 hours.